EVIDENCE THAT RESIDUES -15 TO -46 OF THE HEMOLYSIN SECRETION SIGNAL ARE INVOLVED IN EARLY STEPS IN SECRETION, LEADING TO RECOGNITION OF THETRANSLOCATOR

We previously identified three well-dispersed mutations, E(978)-K, F-9 89-L and D-1009-R within the haemolysin A signal region, located at po sitions -46, -35 and -15, with respect to the C-terminus, respectively . Each mutation reduces the efficiency of secretion two- to threefold leaving 30-45% of the wild-type activity. We have constructed by in vi tro manipulations double mutants of HlyA carrying all combinations of these mutations and a triple mutant carrying all three mutations. The effects on secretion were determined and the results, including residu al levels of secretion with the triple mutant of only 0.6%, compared w ith the wild type, indicated that these residues may interact to form a single function in the wild-type signal. To test this further, we de veloped a secretion competition assay in order to classify signal muta tions. We demonstrated that a CIZ-HlyA fusion protein, containing the C-terminal 81 kDa of HlyA fused to virtually the whole LacZ protein, s trongly inhibits the secretion of the wild-type HIyA co-expressed in t he same cell. The properties of the fusion indicate that it blocks the translocator. The three mutations singly and in combinations were rec ombined in vitro into the 3'-end of the hybrid gene. In every case, th e presence of a mutation in the secretion signal of the hybrid protein alleviated the inhibition of secretion of the co-expressed HIyA. All the mutations are therefore essentially recessive and we propose that they ail affect an early function, probably recognition of the translo cator, rather than a subsequent step involved in translocation or fina l release of the toxin to the medium. This would indicate that residue s involved in recognition (or steps leading to recognition) extend fro m at least -15 to -46 with respect to the HIyA C-terminus.